Preparation
of DNA Template For Direct
Sequencing of Large Insert PAC and BAC
Plasmids*

This protocol is designed to allow the use of 96-well trays
(8x12 microtiter tray format) and multi-channel devices from
the initial inoculation step to the final cycle sequence reaction.
It will yield approximately 200ng of purified plasmid per
ml of culture fluid. When using one of the accompanying
sequencing methods, one cycle sequence reaction will require
160-300ng of template.

1. Streak clone stock to single colony on LB (+antibiotic)
plate. Inoculate 1ml LB (+antibiotic) culture with single
colony, grow overnight at 37°C. Using 3 ul of (overnight growth)
single colony culture fluid, inoculate a single or multiple
96 deep-well blocks containing 1.2-1.6 ml per well of LB media
with appropriate antibiotic. Alternatively, 24 deep-well or
48 deep-well blocks may be used with up to 4.8 or 2.4ml of
media respectively. Cover each block with an AirPore
(Qiagen Inc.) sheet and incubate at 37°C with shaking at 325
RPM for approximately 20 hours.

2. Pellet cells by centrifugation for 20 at 3,200 RPM
in a Sorvall RT7 at 4°C with RTH-250 rotor, ~1700 xg. Decant
and drain pellets. If duplicates are used, transfer culture
fluid from the duplicate block to the first block. Spin again,
decant and drain.